Human Blood Fractions Research Articles

Peripheral human CD5+ and CD5- B cells may express somatically mutated VH5- and VH6-encoded IgM receptors.

Previous studies have indicated that a sizable fraction of adult human peripheral blood B cells may express IgM receptors encoded by somatically mutated V regions. From these studies it was uniquely associated with the peripheral blood B cell compartment, was uniquely associated with the peripheral blood B cell compartment, associated with particular VH gene segments and/or B cell subpopulations. We have addressed these issues by analyzing > 80 VH5 and VH6-encoded mu transcripts from unseparated peripheral blood, tonsil, and spleen B cells, as well as from B cells separated on the basis of CD5 Ag expression. The results demonstrate that somatically mutated VH5 and VH6 regions are ubiquitously expressed in IgM-bearing B cells in all peripheral adult human lymphoid organs, and that the occurrence of somatic mutations does not segregate with either CD5+ or CD5- B cell populations. The distribution and nature of mutations, as well as the occurrence of clonally related but divergent transcripts suggests that at least some of the mutations were selected by Ag.

Effects of activated eosinophils cultured from human umbilical cord blood on guinea pig trachealis.

We studied the biochemical indexes and corresponding induction of airway smooth muscle contraction and hyperresponsiveness in guinea pig trachealis in situ caused by cultured eosinophils derived from mononuclear cell fractions of human umbilical cord blood. A method was developed that permitted isolation of large numbers of cells (approximately 2.6 x 10(6)/ml cord blood) having morphological and immunohistological characteristics of human peripheral blood eosinophils. After activation with 10(-6) M formyl-Met-Leu-Phe + 5 micrograms/ml cytochalasin B (fMLP + B), in situ application to the epithelial surface of 6 x 10(6) cord-derived eosinophils (CDE)/surface area (cm2) caused 1.46 +/- 0.24 g/cm maximal active tracheal tension in guinea pig tracheal smooth muscle (P < 0.005 vs. zero baseline). Muscarinic responsiveness also was augmented in situ in trachealis preparations treated with activated 3-wk CDE. Contraction caused by 3 x 10(-7) mol/kg iv methacholine (MCh) was 0.94 +/- 0.18 g/cm at baseline vs. 1.80 +/- 0.24 g/cm after activated CDE (P = 0.02). Control (sham-activated) 3-wk CDE caused neither significant contraction [0.41 +/- 0.16 g/cm active tension (AT); P < 0.05 vs. fMLP+B] nor augmented muscarinic responsiveness. Cells cultured for 5 wk contained fewer granules than 3-wk CDE and also caused less direct contraction of trachealis (0.73 +/- 0.14 g/cm AT) after activation (P < 0.01 vs. 3-wk CDE). Both contraction and muscarinic augmentation were blocked in 3-wk CDE after blockade of leukotriene C4 (LTC4) synthesis by pretreatment with the 5-lipoxygenase inhibitor, A63162 (50 microM). Treatment with A63162 had no effect on the stimulated release of eosinophil peroxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases.

To estimate the extent of genomic DNA damage and killing of lymphocytes by reactive oxygen intermediates in autoimmune diseases. 8-Oxo-7-hydrodeoxyguanosine (8-oxodG), a promutagenic DNA lesion induced by reactive oxygen intermediates, was measured by high performance liquid chromatography, coupled with electrochemical detection, in hydrolysates of DNA which had been extracted from lymphocyte and polymorphonuclear leucocyte fractions of human blood. In addition, human primary blood lymphocytes stimulated by concanavalin A were assayed for cytotoxicity induced by hydrogen peroxide on day 0, by assessing cell proliferation during seven days of culture. Constitutive 8-oxodG was detectable (mean (2 SEM) moles 8-oxodG/10(6) moles deoxyguanosine) in DNA isolated from normal human blood lymphocytes (68 (8), n = 26) and polymorphonuclear leucocytes (118 (24), n = 24). Lymphocyte DNA from donors with the following inflammatory autoimmune diseases contained significantly higher levels of 8-oxodG than that from healthy donors: rheumatoid arthritis (98 (16)), systemic lupus erythematosus (137 (28)), vasculitis (100 (32)), and Behçet's disease (92 (19)). Lymphocyte 8-oxodG levels in non-autoimmune controls and patients with scleroderma were not significantly different from those of healthy controls. The levels of 8-oxodG were significantly higher in the DNA from normal polymorphonuclear leucocytes than in paired DNA samples from normal lymphocytes, but there were no differences between levels of 8-oxodG in polymorphonuclear leucocytes from normal subjects and the patients studied. Levels of 8-oxodG did not correlate with disease duration, disease severity, or age. Lymphocytes from patients with systemic lupus erythematosus and rheumatoid arthritis, but not those with scleroderma, also showed cellular hypersensitivity to the toxic effects of hydrogen peroxide. There was increased genomic DNA damage, and increased susceptibility to cytotoxic killing by hydrogen peroxide, in lymphocytes from patients with certain autoimmune diseases. These results might be explained by defective repair of DNA damage or by increased production of reactive oxygen intermediates in inflammation. Although more direct studies are needed, the evidence available favours the former explanation.

Binding of 5-Methoxypsoralen to Blood Fractions

The binding of 5-methoxypsoralen (5-MOP) to human serum and blood fractions was studied by equilibrium dialysis associated to high-performance liquid chromatography. 5-MOP serum binding was 95% and kept constant in the range of therapeutic concentrations. Albumin was the main binding protein with one class of binding sites (n = 2.05) and with a moderate affinity constant (K&lt;sub&gt;a&lt;/sub&gt; = 10,270 &lt;i&gt;M&lt;/i&gt;&lt;sup&gt;-1&lt;/sup&gt;). Free fatty acids (FFA) enhanced 5-MOP binding to albumin. Binding to other proteins, α&lt;sub&gt;1&lt;/sub&gt;-acid glycoprotein, gamma-globulins and lipoproteins (LDL, HDL, VLDL) also occurred, but was negligible. 5-MOP bound to red blood cells (RBCs) with a binding index (ratio of intra- to extra-globular 5-MOP concentrations) of 3.84 and 1.48 in buffer and plasma, respectively. 5-MOP distribution in blood was simulated. The free fraction of 5-MOP in blood could be increased in hypoalbuminemia, unchanged in acute inflammation, and decreased by FFA.

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

Effects of Synergistic Cytokine Combinations, Low Oxygen, and Irradiated Stroma on the Expansion of Human Cord Blood Progenitors

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL-1/IL-3. This indicates that an earlier cell type was affected by IL-3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL-6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

Generation of monoclonal antibodies against soluble human T cell receptor polypeptides

One approach to the diagnosis and therapy of T cell-mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the "phosphatidylinositol linkage" strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcR alpha and beta chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti-beta chain mAb, denoted anti-V beta 13, stain a significant (approximately 5%) fraction of human peripheral blood alpha/beta T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activate T cells transfected with the relevant alpha and beta chain cDNA. Anti-alpha chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.

A large fraction of human peripheral blood gamma/delta + T cells is activated by Mycobacterium tuberculosis but not by its 65-kD heat shock protein.

We report that M. tuberculosis organisms, but neither PHA nor allogeneic stimulator cells, preferentially activate gamma/delta+ cells within E rosette-purified peripheral blood T cells. gamma/delta+ T cells from purified protein derivative (PPD)-nonimmune healthy donors were enriched by depletion of CD4+ and CD8+ cells; double-negative (DN) cells contained 65-92% gamma/delta+ T cells. Limiting dilution (LD) analyses revealed that 1 of 2-19 purified DN cells proliferated in response to mycobacteria, while frequencies of DN cells proliferating in response to a recombinant 65-kD heat shock protein (hsp 65) of M. tuberculosis/M. bovis were 10-20-fold lower. Established clones of mycobacteria-reactive gamma/delta+ T cells specifically recognized mycobacteria, but neither PPD nor hsp 65. Restimulation of these clones required the presence of PBMC feeder cells; EBV-transformed lymphoblastoid cell lines could not substitute for PBMC. Mycobacteria-reactive gamma/delta+ clones proliferated equally well in the presence of autologous or allogeneic (HLA-DR-different) PBMC feeder cells and thus were not MHC class II restricted. Taken together, these results demonstrate that mycobacteria-reactive gamma/delta+ T cells are present in high frequency in the peripheral blood of healthy individuals, and suggest that hsp 65 of mycobacteria is not a major antigen for gamma/delta+ T cells of normal PPD-nonimmune blood donors.

Influence of a plasma fraction of human blood, rich in high density lipoprotein, on in vitro formation of prostaglandin I 2 (PGI 2) and thromboxane A 2 (TXA 2)

The influence of a plasma fraction of human blood, rich in high density lipoprotein (HDL), was investigated on the “in vitro” formation of PGI 2 and TXA 2. The addition of 1 mg HDL-cholesterol per ml incubation fluid stimulated significantly the biotransformation of prostaglandin H 2 into PGI 2 by the microsomal fraction of pig aorta. The TXB 2 formation capacity of whole clotted blood was inhibited by administration of HDL in a dose dependent manner. These results suggest that added HDL is able to enhance the ratio PGI 2:TXA 2. This did not depend on the preparation of HDL either by ultracentrifugation or by precipitation.

Effects of ethanol, acetaldehyde and disulfiram on the metabolism of biogenic aldehydes in isolated human blood cells and platelets

The effects of ethanol, acetaldehyde and disulfiram on the metabolism of biogenic aldehydes were measured in different human blood fractions. Intact erythrocytes, leukocytes and platelets were incubated in phosphate-buffered saline with 3,4-dihydroxyphenylacetaldehyde (DOPAL) or 5-hydroxyindole-3-acetaldehyde (5-HIAL), the aldehydes derived from dopamine and serotonin, respectively. The disappearance of the aldehyde and the formation of acid and alcohol metabolites were analysed in the presence of different concentrations of ethanol, acetaldehyde or disulfiram using high-performance liquid chromatography with electrochemical detection. Ethanol at a concentration of 20 mM did not affect the biogenic aldehyde metabolism. High concentrations of acetaldehyde caused a dose-dependent inhibition of the disappearance rate of the biogenic aldehydes and of the formation rate of acid metabolites. In incubations with leukocytes or platelets, the inhibition of the acid formation was associated with a slight increase in the formation of the alcohol metabolites. Disulfiram at a concentration of 50 μM totally inhibited the metabolism of DOPAL and 5-HIAL in incubations with erythrocytes or platelets, whereas much less inhibition was observed in incubations with leukocytes.

A rapid method for the preparation of the neutrophil fraction of granulocytes from human blood by centrifugation on isotonic Nycodenz gradients.

A rapid method for the preparation of neutrophils of the granulocyte fraction of human blood is described. A leucocyte-rich fraction is loaded onto preformed isotonic Nycodenz gradients, formed by mixing a stock isotonic Nycodenz solution with a NaCl diluent solution. After low speed centrifugation a neutrophil fraction of high viability, yield and purity can be isolated from the gradients.

Metabolism of biogenic aldehydes in isolated human blood cells, platelets and in plasma

The metabolism of biogenic aldehydes was measured in different human blood fractions. Isolated erythrocytes, leukocytes, platelets and plasma were incubated with 3,4-dihydroxyphenyl-acetaldehyde (DOPAL) or 5-hydroxyindole-3-acetaldehyde (5-HIAL), the aldehydes derived from dopamine and 5-hydroxytryptamine, respectively. The disappearance of the aldehydes and the formation of acid and alcohol metabolites were analysed using high-performance liquid chromatography with electrochemical detection. The aldehydes were unstable in phosphate-buffered saline, but this non-enzymatic oxidation was prevented in the presence of EDTA, pyrophosphate or blood tissue. When DOPAL or 5-HIAL were incubated with erythrocytes, only acid metabolites were formed, whereas both acid and alcohol metabolites were formed in incubations with leukocytes or platelets. The amount of the acid metabolite exceeded that of the alcohol metabolite, both with leukocytes and platelets. No metabolites were formed when the aldehydes were incubated in plasma. The oxidation of the aldehydes in incubations with erythrocytes or platelets was totally inhibited in the presence of 50 μM of the aldehyde dehydrogenase inhibitor disulfiram. However, disulfiram did not inhibit the metabolism of DOPAL and 5-HIAL in incubations with leukocytes, suggesting that different isozymes of aldehyde dehydrogenase are present in leukocytes as compared to erythrocytes and platelets.

Marked activation of benzo[a]pyrene to protein-binding forms in the presence of unsaturated fatty acids and heme-compounds.

In vitro activation of benzo[a]pyrene to protein-binding forms was investigated with the following two systems: (1) various fractions of human and rat blood had a potential of activating benzo[a]pyrene to forms binding to proteins in the presence of linoleic or arachidonic acid. Red blood cells activated much more benzo[a]pyrene than platelets or lymphocytes. Variation in the individual activity of the respective cell types and the relative activity between cells from different individual donors was observed; and (2) more prominent activation of benzo[a]pyrene was observed with a simplified system, in which a combination of unsaturated fatty acid, i.e., linoleic or arachidonic acid, and heme-compounds resulted in activation of benzo[a]pyrene. Although the binding of benzo[a]pyrene to bovine serum albumin did increase with incubation time, an initial rapid reaction within the first few minutes after addition of the substrates was followed by very much reduced rates of binding thereafter. Hematin and hemin could be utilized for the activation of benzo[a]pyrene, but biliverdin, hematoporphyrin and protoporphyrin IX could not. The present results suggest a possible role for unsaturated fatty acids and heme-compounds in the activation of benzo[a]pyrene in vivo.

Expression of the Mammalian c-fes Protein in Hematopoietic Cells and Identification of a Distinct fes-Related Protein

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

Transport of conjugates of toxicants by blood proteins

Binding of naphthol and its glucoside and glucuronide conjugates by blood proteins was studied in vitro and in vivo. Binding was found to be primarily to the albumin fraction of human blood and the binding constants were moderate to low. Both in vivo (mice) and in vitro (human) experiments suggest that a substantial portion of naphthol and two conjugates are transported in bound form to the site of elimination.

Morphine diesters. II. Blood metabolism and analgesic activity in the rat.

The kinetics of hydrolysis of dipropanoylmorphine (DPM) and dibutanoylmorphine (DBM) in human blood fractions and for diacetylmorphine (DAM) and DBM in rat blood fractions were investigated. In each case the hydrolysis of morphine diesters terminated with the production of the corresponding 6-monoester derivative. Generally, decreases in Km and Vmax were observed for the plasma, red blood cell (RBC) cytosol, and RBC membrane esterases responsible for morphine diester hydrolysis as the alkyl chain length of the ester moiety increased. This resulted in an overall decrease in the rate of hydrolysis of morphine diesters by human or rat blood with longer chain homologs of DAM. The analgesic potency and duration of morphine, DAM, and DBM were assessed at various i.v. dosages in the rat by means of the tail-flick latency test. A comparison of equianalgesic doses of morphine, DAM, and DBM indicated that DAM and DBM were 11.5 and 6 times as potent and 0.8 and 1.2 times as long acting, respectively, as morphine.

Characteristics of 2,4,5,2′,4′,5′-hexachlorobiphenyl distribution among lipoproteins in vitro

The uptake, distribution, and transfer of 2,4,5,2′,4′,5′-hexachlorobiphenyl (6-CB) were examined in vitro with human and rat whole blood, plasma, and lipoprotein fractions. 6-CB distribution between plasma and erythrocytes as well as among lipoproteins was determined following sedimentation of erythrocytes and ultracentrifugal fractionation of plasma. In both rat and human whole blood, 70 to 75% of 6-CB partitioned into plasma and 25 to 30% into erythrocytes. The uptake of 6-CB into plasma was extremely rapid and the rate of uptake was found to be dependent upon temperature. The distribution of 6-CB among lipoproteins was relatively homogeneous with 20 to 30% being distributed in very low-density lipoproteins (VLDL, d = 0.95 – 1.006 g/ml), 15 to 20% in low-density lipoproteins (LDL, d = 1.006 – 1.063 g/ml), and 15 to 25% in high-density lipoproteins (HDL, d = 1.063 – 1.21 g/ml). Over 25% of 6-CB was found in the remaining bottom fraction. In addition, each isolated fraction when incubated alone with 6-CB was shown capable of uptake. The relative proportion of 6-CB among the lipoproteins was independent of the level taken up by plasma. 6-CB was also found to transfer among lipoproteins. This exchange of 6-CB proved to be dependent upon the concentrations of both protein and triacylglycerol in the incubations. Two proteins in the bottom fraction (Bf), albumin and a steroid binding globulin, were capable of competing with the lipoproteins for 6-CB uptake.

Diacetylmorphine (heroin) hydrolases in human blood.

Human blood esterases responsible for diacetylmorphine (DAM, heroin) metabolism were examined by means of an HPLC assay system for DAM, 6-monoacetylmorphine (MAM), and morphine. Four kinetically distinct enzymes capable of hydrolyzing DAM to MAM were observed in the plasma, erythrocyte (RBC) cytosol, and RBC membrane fractions of human blood. Under physiological conditions (pH 7.4, 37 degrees C), the Km (micromolar) and Vmax (micromolar per minute) values, respectively, for these enzymes were as follows: plasma, 200 and 210; RBC cytosol, 710 and 815; RBC membranes, 2000 and 748, and 38 and 80. For DAM blood concentrations less than or equal to 100 ng/mL, RBC associated esterases are responsible for the majority (76%) of DAM hydrolysis in blood.